Methods and apparatuses for conducting assays

ABSTRACT

Disclosed are methods for conducting assays of samples, such as whole blood, that may contain cells or other particulate matter. Also disclosed are systems, devices, equipment, kits and reagents for use in such methods. One advantage of certain disclosed methods and systems is the ability to rapidly measure the concentration of an analyte of interest in blood plasma from a whole blood sample without blood separation and hematocrit correction.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/831,139,filed Jul. 6, 2010, now pending, which is a division of U.S. applicationSer. No. 11/145,528, filed Jun. 3, 2005 and now patented as U.S. Pat.No. 7,776,583 granted on Aug. 17, 2010, which claims the benefit ofpriority to: U.S. Provisional Patent Application Ser. No. 60/576,710,filed Jun. 3, 2004, titled METHODS AND APPARATUSES FOR CONDUCTINGASSAYS, by M. Billadeau, et al., all of which are incorporated herein byreference.

FIELD OF THE INVENTION

The present invention is directed to methods for conducting assays ofsamples, such as whole blood, that may contain cells or otherparticulate matter. The invention also relates to systems, devices,equipment, kits and reagents for use in such methods.

BACKGROUND OF THE INVENTION

Traditionally, clinical measurements (especially, solid phase bindingassays) for analytes in blood have been carried out in serum or plasmasamples derived from the blood. Partly for this reason, clinicianspresent the levels of most blood markers in terms of the concentrationof the marker in the liquid fraction of the blood sample (e.g., theconcentration in plasma or serum fractions derived from the bloodsample). Assays carried out in whole blood have had limited acceptance,in part because conventional assays give different signals for a wholeblood sample relative to a plasma or serum fraction derived from thesame sample. This difference is primarily due to the difference inconcentration of the analyte in the different sample types: theconcentration of an analyte in a whole blood sample being effectivelydiluted relative to the concentration of the analyte in the liquidfraction because of the volume occupied by the red blood cells. Thedifference in signals makes it difficult to compare results to wellestablished reference ranges that have been expressed in terms of theconcentration of the analyte in serum or plasma. In addition, thepresence of a large volume fraction of cells in a whole blood sample mayalso interfere with many assay technologies and make it difficult tocarry out precise and accurate measurements.

Piironen T, et. al., (2001) Clinical Chem., 74(4): 703-711 andTarkkinen, P., et al., (2002) Clinical Chem., 48(2) 269-277) disclosed,respectively, immunoassays of whole blood samples for prostate specificantigen and C-reactive protein (CRP). Both references report correlationcurves comparing the measured levels of the analytes in whole bloodrelative to the measured levels in the corresponding plasma or serumfractions. In both cases, the authors reported that the graphs could befit by lines with slopes of roughly 0.56-0.57, the difference from unitybeing attributed to the average hematocrit (the relative volume of bloodoccupied by erythrocytes) of the whole blood samples. The authorspropose correcting measurements in whole blood samples using theassumption that the samples have a hematocrit value equal to the averagehematocrit for the patient population. Because of the wide range forhematocrit values that may be observed in patient samples (the normalrange is from 41-50% for adult males and 35-46% for adult females, buthematocrit levels of less than 20% can be observed in severe cases ofanemia), this approach could lead to large errors in the reportedvalues.

U.S. Pat. No. 6,475,372 to Ohara T J et al.; U.S. Pat. No. 6,106,778 toOku N. et al. disclose apparatuses for measuring both the concentrationof an analyte in a whole blood sample and the hematocrit of the sample.The measured concentration value is converted to the concentration ofanalyte in the liquid fraction by using the measured hematocrit value toapply a hematocrit correction. This approach adds complexity to theassay apparatus and may be subject to increased imprecision because theerror is a function of the variability in both the assay signal andhematocrit determination.

Other approaches developed for conducting assays on whole blood sampleshave used instrumentation that employ integrated filters (includinglateral flow membranes) or centrifuges to provide for separation of thered blood cells from plasma or serum fractions prior to the measurementof the analyte. While these approaches avoid the need for a hematocritcorrection, they add significant cost and complexity to theinstrumentation. In addition, filtration has the disadvantages ofpossible loss of analyte to the filter material and limitations in thesample volume that can be easily processed.

SUMMARY OF THE INVENTION

The invention relates to methods for conducting assays for measuringanalytes in samples, which samples may contain particulate matter, andrelated apparatus. The invention includes methods that permit themeasurement of the concentration of an analyte in the liquid fraction ofthe sample. This measurement may be carried out without removing theparticulate matter from the sample and/or without correcting the resultsto account for the volume of the sample occupied by the particulatematter. In many cases, the measurement of the concentration of ananalyte in the liquid fraction of the sample performed according tocertain inventive methods, e.g. without prior removal of the particulatematter from the sample and/or without correcting the results to accountfor the volume of the sample occupied by the particulate matter, closelyapproximates, nearly equals, or equals the actual concentration of theanalyte in the liquid fraction of the contiguous sample (i.e. as wouldbe measured for a sample consisting of the pure liquid fraction fromwhich essentially all particulate matter was removed prior to themeasurement). In certain embodiments, the concentration of the analytein the sample as measured using the inventive methods will differ fromthe actual concentration of the analyte in the liquid fraction of thesample by no more than 20%, 10%, 5%, 2%, 1%, 0.5%, or even less.According to one embodiment, samples differing greatly in the volumefraction occupied by particulate matter may be analyzed withoutmeasuring or requiring knowledge of the value of the volume fraction.The invention also relates to systems, devices, equipment, kits andreagents for use in such methods.

One embodiment of the invention relates to a method for measuring one ormore analytes of interest comprising exposing a sample containingparticulate matter to a surface, such as a binding surface, so that atleast a portion of the sample, contacts the surface, optionallyimmobilizing an amount of an analyte on the surface, and measuring theamount of analyte on the surface. For example, a portion consisting ofor consisting essentially of the liquid fraction of the contiguousvolume of a sample may contact the surface. The measured amount isdependent on the concentration of the analytes in the liquid fraction ofthe sample and is substantially independent of the volume occupied byparticulate matter in the sample.

Another embodiment of the invention relates to a method for measuringone or more analytes of interest comprising exposing a sample containingparticulate matter to a surface, such as a binding surface, so that atleast a portion of the sample contacts the surface, optionallyimmobilizing an amount of an analyte on said surface and measuring theamount of the analyte immobilized on the surface, and determining aconcentration of the analyte in the sample that differs from the actualconcentration of the analyte the liquid fraction of the sample by nomore than 20%, 10%, 5%, 2%, 1%, or 0.5%. For example, a portionconsisting of or consisting essentially of the liquid fraction of thecontiguous volume of a sample may contact the surface. The determinationis achieved without correcting for the volume of the sample occupied byparticulate matter.

Yet another embodiment of the invention relates to a method formeasuring one or more analytes of interest comprising exposing a samplecontaining particulate matter to a surface, such as a binding surface,so that at least a portion of the sample contacts the surface,immobilizing an amount of an analyte on the surface and generating anassay signal that is indicative of the amount of the analyte immobilizedon the surface. For example, a portion consisting of or consistingessentially of the liquid fraction of the contiguous volume of a samplemay contact the surface. The assay signal is dependent on theconcentration of the analyte in the liquid fraction of the sample and issubstantially independent of the volume occupied by particulate matterin the sample.

Yet another embodiment of the invention relates to a method formeasuring one or more analytes of interest comprising exposing a samplecontaining particulate matter to a surface, such as a binding surface,so that at least a portion of the sample contacts the surface,immobilizing an amount of an analyte on the surface, generating an assaysignal that is indicative of the amount of the analyte immobilized onthe surface and determining, from the measured amount, a concentrationof the analyte in the sample that differs from the actual concentrationof the analyte the liquid fraction of the sample by no more than 20%,10%, 5%, 2%, 1%, or 0.5%. For example, a portion consisting of orconsisting essentially of the liquid fraction of the contiguous volumeof a sample may contact the surface. The determination is achievedwithout correcting for the volume occupied by particulate matter.

Yet another embodiment of the invention relates to a method formeasuring one or more analytes of interest comprising determining, in aparticle-containing fluid sample comprising a fluid fraction and aplurality of particles suspended therein and containing the analyte, ameasurement of the concentration of the analyte present in the fluidfraction that is substantially independent of the volume of theparticles suspended in the fluid sample.

The assay methods of the invention are suitable for measuring the plasmaconcentration of analytes in whole blood samples without a separate stepof separating the red blood cells and/or without correcting forhematocrit. In certain embodiments, a solid phase binding assay fordetermining the plasma concentration is used. In certain suchembodiments of solid phase binding assays of the invention, the amountof an analyte immobilized on a binding surface and any correspondingassay signal indicative of this amount, is dependent on the plasmaconcentration of analyte in a whole blood sample and is independent ofthe hematocrit of the sample. In many cases, the measurement of theplasma concentration of the whole blood sample performed according tocertain inventive methods, e.g. without prior removal of the blood cellsfrom the sample and/or without correcting the results to account forhematocrit, closely approximates, nearly equals, or equals the actualplasma concentration of the analyte (i.e. as would be measured for asample consisting of the pure plasma fraction from which essentially allblood cells were removed prior to the measurement). In certainembodiments, the concentration of the analyte in the sample measuredusing the inventive methods will differ from the actual plasmaconcentration of the analyte by no more than 20%, 10%, 5%, 2%, 1%, 0.5%,or even less.

In certain assay methods of the invention, the act of contacting atleast a portion of a sample with a binding surface may comprisecontacting the sample with a plurality of binding domains on one or morebinding surfaces, where the binding domains have different specificityfor analytes of interest. Accordingly, the assay methods may furthercomprise measuring one or more additional analytes in the sample. In oneembodiment, each analyte of interest is immobilized in a differentbinding domain and the amount of each analyte in the correspondingbinding domain is measured, e.g., by measuring an assay signalindicative of the amount of immobilized analyte.

In certain embodiments, the immobilization is performed over an intervalof time, for example an interval of time less than 10 minutes, or lessthan 5 minutes. During this interval of time, only a fraction of theanalyte present in the sample may be, immobilized. In certainembodiments, less than 30% of an analyte, less than 20% of an analyteand less than 10% of an analyte in a sample is immobilized on thebinding surface.

The assay methods of the invention may include calibrating the assayusing calibrator samples with known concentrations of said analyte.These calibrator samples may be free of particulate matter. For example,in the case of assays suitable for use with whole blood samples, thecalibrator samples may be substantially free of red blood cells.

The assay methods may employ a variety of assay formats includingsandwich assay and/or competitive assay formats.

The binding surfaces (or binding domains within a binding surface) maycomprise a binding reagent immobilized thereon. This binding reagent maybind to an analyte of interest. The binding of the analyte of interestto the binding reagent on the surface may be direct or may occur via oneor more bridging reagents. Accordingly, the assay methods of theinvention may include contacting the sample with a bridging reagent thatbinds both the binding reagent immobilized on the binding surface and ananalyte.

The assay methods of the invention may further comprise contacting ananalyte of interest with i) a labeled binding reagent that binds theanalyte and/or ii) a labeled competitor of the analyte. The bindingreagent or competitor, may be labeled with a label such as, withoutlimitation, ECL labels, luminescent labels, fluorescent labels,phosphorescent labels, radioactive labels, enzyme labels, electroactivelabels, magnetic labels and light scattering labels.

The binding surfaces used in the solid phase binding assay methods ofcertain embodiments of the invention may be surfaces within a flow cell.The binding surfaces may include one or more electrode surfaces, whichmay be located within a flow cell. The binding surfaces used in themethods of the invention may be rough. In certain embodiments, thesurfaces are sufficiently rough so that the surface area accessible toan analyte is at least two-fold larger than the surface area accessibleto red blood cells.

The solid phase binding assay methods of certain embodiments of theinvention may include flowing the sample over the binding surface. Theflowing act may include flowing the sample over the binding surface in aback and forth motion. In certain embodiments, the flow is laminarand/or is characterized by Reynold's numbers of less than 100, or, incertain embodiments, of less than 10. In certain embodiments of theinvention, the flow of whole blood samples over a binding surface iscarried out under conditions that provide a plasma-rich layer adjacentto the binding surface. In certain embodiments, the binding is carriedout under conditions that do not deplete analyte from the sample.

In one particular embodiment such a method comprises creating a flow ofa contiguous volume of a whole blood sample over a surface; therebysegregating blood cells contained in the sample away from the surface tocreate a first, plasma rich, region of the sample in contact with thesurface and a second, cell rich, region of the sample separated from thesurface by the first region, wherein the concentration of blood cells inthe second region substantially exceeds the concentration of blood cellsin the first region; and determining a concentration of an analyte ofinterest in fluid present in the first, plasma enriched, region. Inanother particular embodiment a method comprises acts of: (1) creating aflow of a whole blood sample, for example a contiguous volume of a wholeblood sample, over a surface, wherein the blood flow segregates bloodcells contained in the sample away from the surface to create a plasmaenriched first region of the sample; and (2) determining a concentrationof an analyte of interest in first region. A skilled artisan may readilyrecognize that the invention encompasses embodiments where theparticulate (e.g. blood cell) concentration in the contiguous volume ofa sample exists as a gradient, which may be a continuous gradient, overthe cross-section of the flow channel; in such cases, the first and thesecond regions may comprise spatial designations representative of firstand second fractions of the contiguous volume of the sample that havedifferent volume-averaged particulate (e.g. blood cell) concentrations.For example, in one embodiment, a contiguous volume of a whole bloodsample flowing in a flow cell of one embodiment of the invention has agradient of blood cell concentration across the entirety of thecontiguous volume of the sample, which gradient is characterized by thepresence of a relatively plasma enriched fraction comprising a firstregion in contact and close proximity with an inner surface of the flowchannel, and relatively cell enriched fraction comprising a secondregion separated from the inner surface by the first region.

In another embodiment, the binding surface is positioned in an assaycell (such as a flow cell) such that, during operation, the surfacefaces sidewise or, downward. Blood cells in a whole blood sample held inthe assay cell can settle to the bottom of the cell and away from thebinding surface so as to provide a plasma-rich layer adjacent thebinding surface. In such an embodiment, the assay methods may includeintroducing a whole blood sample into the cell and allowing the redblood cells to settle. In certain embodiments, the binding is carriedout under conditions that do not substantially deplete analyte from thesample.

In one particular embodiment, a method comprises exposing a whole bloodsample to a surface, for example exposing a contiguous volume of a wholeblood sample to a surface; maintaining blood cells contained in thesample away from at least a portion of the surface to create a first,plasma enriched, region of the sample in contact with the at least aportion of the surface and a second, cell enriched, region of the sampleseparated from the at least a portion of the surface by the firstregion, wherein the concentration of blood cells in the second regionsubstantially exceeds the concentration of blood cells in the firstregion, and determining a concentration of an analyte of interest influid present in the first, plasma rich, region.

The assay methods of the present invention may further comprisedisplacing a sample from a binding surface prior to measuring the amountof one or more analytes immobilized on the surface or prior togenerating an assay signal. Displacement of the sample may be carriedout by introducing a wash solution. The assay methods may even furthercomprise contacting the binding surface with a solution containing alabeled binding reagent or labeled analog of an analyte after displacingthe sample from the binding surface.

The assay methods of the invention may be carried out on undilutedsamples, e.g., undiluted whole blood. In the case of whole bloodsamples, the blood sample may advantageously contain anticoagulants.

One specific embodiment of the present invention relates to a method forperforming a rapid blood test, for example, a clinical diagnostic test,comprising i) drawing blood from a patient to provide a whole bloodsample, ii) applying the whole blood sample to a cartridge having one ormore binding surfaces having one or more binding domains, iii) flowingthe sample over the binding surface(s) for a defined interval of time toimmobilize amounts of one or more analytes, iv) measuring the amounts ofthe one or more analytes immobilized on the binding surface(s), whichmay be immobilized on one or more distinct binding assay domains and v)determining, from the measured amounts, the plasma concentrations of theone or more analytes in the sample. The method may be carried outwithout removing the red blood cells from the sample or correcting forsample hematocrit.

Also discussed are apparatuses that measure the concentration of ananalyte in the liquid fraction of a sample e.g., the plasmaconcentration of an analyte in a whole blood sample). The apparatus maybe configured to carry out the assay methods of the invention. Theapparatus may comprise one or more binding surfaces having one or morebinding domains and, optionally, one or more of the following additionalcomponents: a flow cell comprising the one or more binding surfaces, apump for flowing sample past the binding surface(s), and a detector fordetecting an assay signal.

Also discussed is a kit for measuring the concentration of an analyte inthe liquid fraction of a sample (e.g., the plasma concentration of ananalyte in a whole blood sample). The kit may be, configured to besuitable for use with the assay methods of the invention. The kit maycomprise one or more binding surfaces having one or more binding domainsand, optionally, one or more reagents such as labeled binding reagentsthat bind the analyte, labeled analogs of the analyte, anticoagulants,blocking agents, and pH buffers, etc. These components may be providedin dry form. The kit may also contain liquid components including washbuffers. The kit may further comprise an assay cartridge, which may be adisposable, containing the binding surface(s). The disposable cartridgemay further comprise a flow cell comprising the binding surface(s). Thecartridge may comprise a pump for moving sample over the bindingsurface(s) and/or a detector for detecting an assay signal.Alternatively, one or both of these components may be provided by aseparate cartridge reader apparatus.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are schematic are not intended to be drawn toscale. In the figures, each identical, or substantially similarcomponent that is illustrated in various figures is typicallyrepresented by a single numeral or notation. For purposes of clarity,not every component is labeled in every figure, nor is every componentof each embodiment of the invention shown where illustration is notnecessary to allow those of ordinary skill in the art to understand theinvention. In the drawings:

FIG. 1 shows a schematic representation of the flow of whole blood underconditions that result in the generation of a plasma-rich boundaryregion;

FIG. 2 shows a schematic representation of an assay cartridge accordingto one embodiment;

FIG. 3A shows an exploded view of a cartridge according to oneembodiment;

FIG. 3B shows a detailed view of the alignment of two components of thecartridge of FIG. 3A;

FIG. 4 shows the results of a one-step electrochemiluminescence assayfor myoglobin, TnT, CKMB and TnI in whole blood samples. The plot showsECL signal (vertical axis) for each analyte (horizontal axis) at sixhematocrit levels;

FIG. 5 shows the results of electrochemiluminescence assays formyoglobin, TnT, CKMB and TnI. The plot shows ECL signal (vertical axis)for each analyte (horizontal axis) at various analyte concentrations inblood plasma;

FIG. 6 compares one-step electrochemiluminescence assays for myoglobin,TnT and CKMB in whole blood samples carried out using static incubationconditions or mixing. The plot shows ECL signal (vertical axis) for eachanalyte (horizontal axis) at four hematocrit levels;

FIG. 7 shows the results of a one-step electrochemiluminescence assayfor myoglobin, TnT and CKMB using long incubation times. The plot showsECL signal (vertical axis) for each analyte (horizontal axis) at fourhematocrit levels;

FIG. 8 shows the results of a two-step electrochemiluminescence assayfor TnT and CKMB in whole blood samples. The plot shows ECL signal(vertical axis) for each analyte (horizontal axis) at four hematocritlevels;

FIG. 9 shows the results of one-step and two-stepelectrochemiluminescence assays for progesterone. The plot shows ECLsignal (vertical axis) for each analyte (horizontal axis) at differenthematocrit levels;

DETAILED DESCRIPTION OF THE INVENTION

Disclosed are methods for conducting assays for measuring analytes insamples that may contain particulate matter. Certain embodiments ofinvention involve methods that permit the measurement of theconcentration of an analyte in the liquid fraction of such samples. Thismeasurement may be carried out without the need for prior removal of theparticulate matter from the sample (e.g., without using centrifugation,settling, filtration (e.g., filtration through filters, porous membranesor lateral flow membranes), affinity binding, coagulation, etc. toremove the particulate matter) and/or without correcting the results toaccount for the volume of the sample occupied by the particulate matter.According to one embodiment, samples differing greatly in the volumefraction occupied by particulate matter may be analyzed to achieve ameasure of the concentration of an analyte in the liquid fraction of thesample that can be equal to or closely approximating the actualconcentration in the liquid fraction (i.e. as would be measured for asample consisting of the pure liquid fraction from which essentially allparticulate matter was removed prior to the measurement) withoutmeasuring or requiring knowledge of the value of the volume fractionoccupied by the particles. In certain embodiments, the concentration ofthe analyte in the sample measure using the inventive methods willdiffer from the actual concentration of the analyte in the liquidfraction of the sample by no more 20%, 10%, 5%, 2%, 1%, 0.5%, or evenless.

The methods of certain embodiments of the invention may be used toobtain a measurement of the plasma concentration of an analyte in awhole blood sample. According to one embodiment of the invention, theassay signal for a given plasma concentration of analyte in a wholeblood sample is substantially independent of the hematocrit of thesample and substantially equivalent to the signal obtained for plasma orserum derived from the whole blood sample. Measurement of theconcentration of one or more analytes in the plasma fraction of a wholeblood sample can, therefore, be carried out without requiring themeasurement of the hematocrit or the separation of plasma or serumfractions (e.g., without using centrifugation, settling, filtration,affinity binding, coagulation, etc. to remove the cellular fraction)prior to measurement.

“Measured,” as used herein, may encompass quantitative and qualitativemeasurement, and may encompass measurements carried out for a variety ofpurposes including, but not limited to, detecting the presence of ananalyte, quantitating the amount of an analyte, identifying a knownanalyte, and/or determining the identity of an unknown analyte in asample.

The amount of an analyte in a sample may be presented as a concentrationvalue, i.e., the amount per volume of sample. For samples containingparticulate matter this concentration may be presented as amount pervolume of whole sample, where the volume of whole sample includes thecombined volumes of the liquid and particulate components of the sample.Alternatively, the concentration may be reported in terms of amount pervolume of the liquid fraction or component of the sample.

Whole blood samples contain a liquid fraction (the plasma fraction) anda particulate fraction (a blood cell fraction comprised primarily of redblood cells or RBCs). The relative volumes of these two fractions havetraditionally been measured by centrifuging whole blood and measuringthe hematocrit, the ratio of the volume of the pellet (whichapproximates the volume of the blood cell fraction) to the total volumeof the sample. The concentration of an analyte found in the plasmafraction can be expressed in terms of the whole blood concentration (aterm that refers herein to the amount of analyte per volume of wholeblood in a sample) or the plasma concentration (a term that refersherein to the amount of analyte per volume of plasma in a sample). Thetwo concentrations are related by the following function:C_(WB)=C_(P)(1−H), where C_(WB) is the whole blood concentration, C_(P)is the plasma concentration and H is the hematocrit for the sample.Traditionally, the plasma concentration of an analyte has been measuredby first separating an aliquot of the plasma fraction from the bloodcell fraction by filtration or centrifugation and then measuring theconcentration of analyte in the plasma aliquot. In many clinicalapplications, the concentration of an analyte is presented as a serumconcentration, i.e., the concentration of the analyte in serum derivedfrom a whole blood sample. Serum is prepared by clotting whole blood andremoving the remaining liquid fraction from the clot and cellularcomponents. For analytes present in plasma that are not generated,consumed or bound during the clotting process, the plasma and serumconcentrations are usually roughly equal.

Samples that may be analyzed by the methods of the invention includesamples that contain or may potentially contain a large volume fractionof particulate matter (e.g., a high hematocrit in the case of wholeblood samples). In one embodiment, a sample is analyzed that has, or maypotentially have, a volume fraction of particulate matter (e.g., ahematocrit) between 10 and 80% or between 20 and 80% or between 20% and60%. Examples of samples that may be analyzed include, but are notlimited to, food samples, beverages, samples that comprise suspensionsof dirt, environmental sludges or other environmental samples (such assuspensions of particles filtered, or otherwise concentrated, out of airsamples, water samples, environmental swipes, etc.), and biologicalsamples. Biological samples that may be analyzed include, but are notlimited to, feces, mucosal swabs, physiological fluids and/or samplescontaining suspensions of cells. Specific examples of biological samplesinclude tissue aspirates, tissue homogenates, cell cultures (includingcultures of eukaryotic and prokaryotic cells), urine, cerebrospinalfluid, synovial fluid, amniotic fluid, pleural fluid, pericardial fluid,ascetic fluid and whole blood.

According to one embodiment, the sample is a whole blood sample. Incertain embodiments, the whole blood sample is not diluted prior toanalysis or the dilution kept to a minimum. In certain embodiments, thevolume of a diluted whole blood sample relative to the volume of thewhole blood sample prior to dilution is greater than 50%, or greaterthan 80%, or greater than 90%, or greater than 95%, or greater than 99%or 100% (i.e., the sample is undiluted). Reagents may be added to thewhole blood sample prior to analysis, e.g., binding reagents (such asantibodies), agents that compete with an analyte for binding to abinding reagent, anticoagulants (e.g., heparin, citrate, oxalate, EDTA,etc.), pH buffering components, salts, blocking agents (e.g., proteinsthat block non-specific binding and/or block the binding of heterophileantibodies) and preservatives (e.g., fluoride, iodoacetate, etc.). Thedilution of the whole blood sample may be minimized by adding thesereagents in dry form or in liquid form in a small volume.

Advantageously, the methods of the invention allow the same methodologyand apparatus to be used to measure samples with particulate matter(e.g., whole blood) and samples without particulate matter (e.g., plasmaor serum) without requiring any corrections to be applied to account forthe presence or absence of particles (e.g., a hematocrit correction).The assay methods of the invention may include an act of calibrating anassay by running calibration samples having known concentrations ofanalyte so as to determine the relationship between analyteconcentration and assay signal. This calibration may be carried out bythe end user or by the manufacturer of the assayreagents/kits/consumables/etc. The insensitivity of the assays of theinvention to hematocrit can allow assays for whole blood samples to becalibrated with liquid, cell-free calibration samples, thus eliminatingthe need for storing calibration standards containing red blood cellsand/or applying hematocrit corrections to the calibration parameters.Similarly, the assay methods of the invention may include the act ofrunning one or more control samples to ensure the assay method isperforming within specifications (proper performance being indicated bycontrol signals that fall within pre-determined ranges). Theinsensitivity of the assays of the invention to hematocrit can allow forthe use of cell-free controls for whole blood assays. By analogy,controls and/or calibrators for assays for other particulate containingsamples can be run using particulate-free controls or calibrators.

In certain embodiments, of the invention, the methods include the act ofexposing a sample, for example a contiguous volume of a sample, to asurface comprising an assay reagent and contacting at least a portion ofthe sample, e.g. a liquid fraction rich, particulate poor portion.Optionally, this surface may define, in part, the boundary of acontainer (e.g., a flow cell, well, cuvette, etc.) which holds thesample and/or through which the sample is passed. In one embodiment, theassay reagent comprises a reactant that reacts with an analyte in asample. Suitable reactants include materials that covalently ornon-covalently bind to an analyte or catalysts (e.g., enzymes) thatcatalyze a chemical reaction of an analyte. The method may also comprisemeasuring a signal that results from the chemical reaction, e.g., achange in optical absorbance, a change in fluorescence, the generationof chemiluminescence or electrochemiluminescence, a change inreflectivity, refractive index or light scattering, the accumulation orrelease of detectable labels from the surface, the oxidation orreduction or redox species, an electrical current or potential, changesin magnetic fields, etc.

According to another embodiment of the invention, the methods comprisethe acts of contacting at least a portion of a sample with a bindingsurface, immobilizing an analyte on the surface and measuring theanalyte immobilized on the surface. In one example of such anembodiment, the binding surface is prepared by immobilizing, on asurface, binding reagents that bind the analyte. Optionally, the surfacecomprises an array of binding reagents. Optionally, the surface maydefine, at least in part, the boundary of a container (e.g., a flowcell, well, cuvette, etc.) which holds the sample or through which thesample is passed. The method may also comprise generating an assaysignal that is indicative of the amount of the analyte on the surface,e.g., a change in optical absorbance, a change in fluorescence, thegeneration of chemiluminescence or electrochemiluminescence, a change inreflectivity, refractive index or light scattering, the accumulation orrelease of detectable labels from the surface, the oxidation orreduction or redox species, an electrical current or potential, changesin magnetic fields, etc.

In certain embodiments the extent of a reaction with a reactant on asurface or the amount of an analyte of interest immobilized on a bindingsurface correlates with the concentration of analyte in the liquidfraction of the sample (e.g., the plasma concentration in a whole bloodsample) but is substantially independent of the volume fraction of thesample occupied by particulate material (e.g., the hematocrit of a wholeblood sample). Accordingly, the methods may further comprise the act ofdetermining a measurement of the concentration of the analyte in theliquid fraction of the sample that equals or closely approximates theactual concentration of the analyte in the liquid fraction of the sample(i.e. as would be measured for a sample consisting of the pure liquidfraction from which essentially all particulate matter was removed priorto the measurement). This act may be performed without applying ahematocrit correction. According to one embodiment of the invention, i)the extent of the reaction of an analyte in a whole blood sample with areactant on a surface and/or ii) the amount of an analyte immobilized ona binding surface from a whole blood sample is hematocrit independentand the measurement of this reaction or binding (or assay signalresulting from this reaction or binding) is indicative of the analyteconcentration in the plasma fraction.

Certain embodiments include multiplexed measurements of multipleanalytes in a sample. In one embodiment, the signal generated inresponse to the presence of different analytes has a distinguishablecharacteristic, e.g., wavelength of absorbance, energy of emission,current-voltage relationship, etc. that allows the signals to bedeconvoluted or independently measured. In another embodiment, theanalytes are measured by contacting a sample with assay reagents (e.g.,binding reagents) that are present in spatially segregated assay domains(e.g., binding domains) on one or more surfaces, wherein at least twodomains contain assay reagents that differ in their specificity for theanalytes being measured. The spatial separation of the assay domainsallows for independent measurement of assay signals generated at thedifferent assay domains, e.g., through the wide variety of establishedoptical, magnetic, radioactive and electrochemical techniques that havebeen established for conducting measurements using nucleic acid andprotein arrays. In one specific embodiment, a sample is contacted with asurface comprising an array of assay domains, for example, an array ofbinding domains.

Also provided is a method comprising the act of contacting at least aportion of a sample with one or more surfaces comprising a plurality ofassay domains. Optionally, these surfaces may define, at least in part,one or more boundaries of a container (e.g., a flow cell, well, cuvette,etc.) which holds the sample and/or through which the sample is passed.In one embodiment, the assay domains comprise reactants that react withanalytes in a sample and at least two domains differ in theirspecificity for analytes of interest. Suitable reactants includematerials that covalently or non-covalently bind to an analyte orcatalysts (e.g., enzymes) that catalyzes a chemical reaction of ananalyte. The method may also comprise measuring signals that result fromthe chemical reactions at the different domains, e.g., changes inoptical absorbance, a change in fluorescence, the generation ofchemiluminescence or electrochemiluminescence, a change in reflectivity,refractive index or light scattering, the accumulation or release ofdetectable labels from the domains, the oxidation or reduction or redoxspecies, electrical currents or potentials, changes in magnetic fields,etc.

According to another embodiment of the invention, the methods comprisethe acts of contacting at least a portion of a sample with one or morebinding surfaces comprising a plurality of binding domains, immobilizingone or more analytes on the domains and measuring the analytesimmobilized on the domains. In certain embodiments, at least two of thebinding domains differ in their specificity for analytes of interest. Inone example of such an embodiment, the binding domains are prepared byimmobilizing, on one or more surfaces, discrete domains of bindingreagents that bind analytes of interest. Optionally, the sample isexposed to a binding surface that comprises an array of bindingreagents. Optionally, the surface(s) may define, in part, one or moreboundaries of a container (e.g., a flow cell, well, cuvette, etc.) whichholds the sample or through which the sample is passed. The method mayalso comprise generating assay signals that are indicative of the amountof the analytes in the different binding domains, e.g., changes inoptical absorbance, changes in fluorescence, the generation ofchemiluminescence or electrochemiluminescence, changes in reflectivity,refractive index or light scattering, the accumulation or release ofdetectable labels from the domains, oxidation or reduction or redoxspecies, electrical currents or potentials, changes in magnetic fields,etc.

In certain embodiments, the extent of a reaction of an analyte with areactant in an assay domain or the amount of an analyte of interestimmobilized on a binding domain correlates with the concentration of theanalyte in the liquid fraction of the sample (e.g., the plasmaconcentration in a whole blood sample) but is substantially independentof the volume fraction of the sample occupied by particulate material(e.g., the hematocrit of a whole blood sample). Likewise, the reactionand/or binding of a second analyte at a second domain correlates withthe concentration of the second analyte in the liquid fraction of thesample. Accordingly, the methods may further comprise the act ofdetermining a measurement of the concentration of a plurality ofanalytes in the liquid fraction of the sample that equals or closelyapproximates the actual concentration of the analytes in the liquidfraction of the sample (i.e. as would be measured for a sampleconsisting of the pure liquid fraction from which essentially allparticulate matter was removed prior to the measurement). In certainembodiments, this act does not include applying a hematocrit correction.According to one embodiment of the invention, i) the extent of thereaction of analytes in a whole blood sample with reactants on assaydomains and/or ii) the amount of analytes immobilized on binding domainsfrom a whole blood sample is hematocrit independent and the measurementof these reactions or bindings (or the assay signal resulting from thesereactions or bindings) is indicative of the analyte concentrations inthe plasma fraction.

Analytes that may be measured using the methods of the inventioninclude, but are not limited to proteins, toxins, nucleic acids,microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids,glycoproteins, lipoproteins, polysaccharides, drugs, hormones, steroidsand any modified derivative of the above molecules, or any complexcomprising one or more of the above molecules or combinations thereof.The analytes of interest may be indicative of a disease or diseasecondition.

Another embodiment of the present invention provides a method forobtaining a measurement indicative of the concentration of one or more,e.g., two or more analytes in a plasma fraction of a whole blood sample.Two or more analytes may be measured in the same sample. Panels ofanalytes that can be measured in the same sample include for examplepanels of assays for analytes or activities associated with a diseasestate or physiological conditions. Certain such panels include panels ofcytokines and/or their receptors (e.g., one or more of TNF-α, TNF-β,IL1-α, IL1-β, IL2, IL4, IL6, IL10, IL12, IFN-γ, etc.), growth factorsand/or their receptors (e.g., one or more of EGF, VGF, TGF, VEGF, etc.),drugs of abuse, therapeutic drugs, auto-antibodies (e.g., one or moreantibodies directed against the Sm, RNP, SS-A, SS-B Jo-1, and Scl-70antigens), allergen specific antibodies, tumor markers (e.g., one ormore of CEA, PSA, CA 125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA 21-1, NSE,AFP, etc.), markers of cardiac disease including congestive heartdisease and/or acute myocardial infarction (e.g., one or more ofTroponin T, Troponin I, myoglobin, CKMB, myeloperoxidase, glutathioneperoxidase, β-natriuretic protein (BNP), a-natriuretic protein (ANP),endothelin, aldosterone, C-reactive protein (CRP), etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (β-amyloid,tau-protein, etc.), markers of osteoporosis (e.g., one or more ofcross-linked N or C-telopeptides, total deoxypyridinoline, freedeoxypyridinoline, osteocalcin, alkaline phosphatase, C-terminalpropeptide of type I collagen, bone-specific alkaline phosphatase,etc.), markers of fertility state or fertility associated disorders(e.g., one or more of Estradiol, progesterone, follicle stimulatinghormone (FSH), luetenizing hormone (LH), prolactin, (β-hCG,testosterone, etc.), markers of thyroid disorders (e.g., one or more ofthyroid stimulating hormone (TSH), Total T3, Free T3, Total T4, Free T4,and reverse T3), and markers of prostrate cancer (e.g., one or more oftotal PSA, free PSA, complexed PSA, prostatic acid phosphatase, creatinekinase, etc.). Certain embodiments of invention include measuring, e.g.,one or more, two or more, four or more or 10 or more analytes associatedwith a specific disease state or physiological condition (e.g., analytesgrouped together in the panels listed above).

The binding assays of the invention may employ antibodies as bindingreagents. Other suitable binding reagents for use with the methods ofcertain embodiments of the invention include, but are not limited to,receptors, ligands, haptens, antigens, epitopes, mimitopes, aptamers,hybridization partners, and intercalaters. Suitable binding reagentcompositions include, but are not limited to, proteins, nucleic acids,drugs, steroids, hormones, lipids, polysaccharides, and combinationsthereof. The term “antibody” includes intact antibody molecules(including hybrid antibodies assembled by in vitro re-association ofantibody subunits), antibody fragments and recombinant proteinconstructs comprising an antigen binding domain of an antibody (asdescribed, e.g., in Porter, R. R. and Weir, R. C. J. Cell Physiol., 67(Suppl 1); 51-64 (1966) and Hochman, J. Inbar, D. and Givol, D.Biochemistry 12: 1130 (1973)). The term “antibody” also includes intactantibody molecules, antibody fragments and antibody constructs that havebeen chemically modified, e.g., by the introduction of a label.

One of ordinary skill in the art will be able to readily selectdetection technologies suitable for use with the methods of theinvention. These detection technologies include, but are not limited to,a variety of methods that are currently available for measuringreactions (e.g., for measuring enzymatic reactions or bindingreactions). Some techniques allow for measurements to be made by visualinspection, others may require or benefit from the use of an instrumentto conduct the measurement. Techniques for measuring analytes mayinclude coupling a reaction of the analyte (e.g., an enzyme catalyzedreaction) to a change in optical absorbance, fluorescence,chemiluminescence, electrical current, electrical potential, etc.Techniques available for measuring binding assays include solid phasebinding assay techniques in which binding reaction products are formedon a surface and homogenous binding assay techniques in which bindingreaction products remain in solution. Suitable detection techniques maydetect binding events by measuring the participation of labeled bindingreagents through the measurement of the labels via theirphotoluminescence (e.g., via measurement of fluorescence, time-resolvedfluorescence, evanescent wave fluorescence, up-converting phosphors,multi-photon fluorescence, etc.), chemiluminescence,electrochemiluminescence, light scattering, optical absorbance,radioactivity, magnetic fields, enzymatic activity (e.g., by measuringenzyme activity through enzymatic reactions that cause changes inoptical absorbance or fluorescence or cause the emission ofchemiluminescence). Alternatively, detection techniques may be used thatdo not require the use of labels, e.g., techniques based on measuringmass (e.g., surface acoustic wave measurements), refractive index (e.g.,surface plasmon resonance measurements), or the inherent luminescence ofan analyte.

An immunoassay or other type of specific binding assay according tocertain embodiments of the invention can involve a number of formatsavailable in the art including solid phase binding assay formats. Theantibodies and/or other types of specific binding partners can belabeled with a label or immobilized on a surface. Suitable surfacesinclude, but are not limited to, glass, ceramic, polymer, polymercomposite, and metal surfaces. A variety of different textured surfacesmay be used including flat surfaces and rough surfaces. In oneembodiment, the surface is an electrode surface, e.g., an electrodesurface within a multi-well plate, a flow cell or a flow cell chamber ofa cartridge. Surfaces that are rough and/or suitable for use aselectrodes may be provided for by using a surface that comprises amaterial comprising elemental carbon, for example, a composite materialcontaining particulate carbon in a matrix, e.g., a carbon ink.

In embodiments of the invention that employ a solid phase binding assayformat, the method, may comprise binding an analyte in a sample to abinding reagent (the capture reagent) immobilized on a binding surface(e.g., by contacting at least a portion of the sample with the surface)and measuring the amount of analyte bound to the surface. “Binding,” asused herein, may refer to a direct interaction between a binding reagentand an analyte (e.g., the binding of an analyte to an immobilizedanti-analyte antibody) or may involve an indirect interaction throughone or more intermediate species (e.g., the binding to immobilizedstreptavidin of a complex comprising an analyte bound to abiotin-labeled anti-analyte antibody). These intermediate species may bereferred to herein as “bridging” species. Multiplexed assays may becarried out in an analogous fashion by employing a plurality of bindingdomains comprising capture reagents, the binding domains differing intheir selectivity for analytes of interest. By way of example, amultiplexed method may comprise contacting a sample with a plurality ofbinding domains on one or more surfaces, binding a first analyte to afirst of the plurality of domains, binding a second analyte to a secondof the plurality of domains and measuring the amount of the firstanalyte on the first domain and the second analyte on the second domain.

The solid phase binding assay may employ a sandwich binding assayformat. Such a method may comprise binding an analyte in a sample to abinding reagent (the capture reagent) immobilized on a binding surface,binding the analyte to another binding reagent (the detection reagent)comprising a detectable label and measuring the amount of the detectablelabel on the binding surface. The assay may be carried out underconditions that allow both the first and second binding reagents to bindto the analyte to form a “sandwich” complex. Examples of sandwichimmunoassays performed on test strips are described in U.S. Pat. No.4,168,146 to Grubb et al. and U.S. Pat. No. 4,366,241 to Tom et al.,both of which are incorporated herein by reference. Multiplexed assaysmay be carried out in an analogous fashion by employing a plurality ofbinding domains, on one or more surfaces, comprising capture reagents,the binding domains differing in their selectivity for analytes ofinterest. One or more detection reagents may be used, as necessary, tobind to the different analytes of interest. By way of example, amultiplexed assay may comprise contacting at least a portion of a samplewith a plurality of binding domains on one or more surfaces, binding afirst analyte to a first of the plurality of domains, binding a secondanalyte to a second of the plurality of domains, binding the first andsecond analytes to one or more labeled detection reagents and measuringthe amount of label on the first domain and second domains.

The solid phase binding assay may employ a competitive binding assayformat. One such method comprises a) competitively binding to a bindingreagent (the capture reagent) immobilized on a binding surface i) ananalyte in a sample and ii) a labeled analog of the analyte comprising adetectable label (the detection reagent) and b) measuring the amount ofthe label on the binding surface. Another such method comprises a)competitively binding to a binding reagent having a detectable label(the detection reagent) i) an analyte in a sample and ii) an analog ofthe analyte that is immobilized on a binding surface (the capturereagent) and b) measuring the amount of the label on the bindingsurface. An “analog of an analyte” refers, herein, to a species thatcompetes with the analyte for binding to a binding reagent. Examples ofcompetitive immunoassays are disclosed in U.S. Pat. No. 4,235,601 toDeutsch et al., U.S. Pat. No. 4,442,204 to Liotta, and U.S. Pat. No.5,208,535 to Buechler et al., all of which are incorporated herein byreference. Multiplexed assays may be carried out in an analogous fashionby employing a plurality of binding domains, on one or more surfaces,comprising capture reagents, the binding domains differing in theirselectivity for binding or competing with analytes of interest. One ormore detection reagents may be used, as necessary, to bind or competewith the different analytes of interest. By way of example, amultiplexed assay may comprise contacting a sample with a plurality ofbinding domains on one or more surfaces and contacting the bindingdomains with one or more labeled detection reagents, wherein the bindingof a first detection reagent to a first binding domain is competitivelyinhibited by a first analyte and the binding of a second detectionreagent to a second binding domain is competitively inhibited by asecond detection reagent.

By appropriate selection of capture and detection antibodies,multiplexed solid phase binding assays may be carried out that includeboth sandwich binding assays and competitive binding assays.

The solid phase binding methods of certain embodiments of the presentinvention may further comprise displacing (e.g., washing or otherwiseremoving) sample from a binding surface prior to measuring the amount ofbound analyte. Displacement may be effected, e.g., by washing thesurface with a wash reagent or by displacing sample material with air.In some embodiments of the invention that include a displacement act,the surface is, concurrently or subsequent to the displacement act,contacted with a detection reagent. By way of example, in one embodimentof a sandwich binding assay that includes a displacement act, at least aportion of a sample is contacted with a binding surface so as to captureanalyte on the surface, the sample is removed, and the surface iscontacted with a labeled binding reagent that binds to captured analyteand forms the sandwich complex. In one embodiment of a competitive assaythat includes a displacement act, at least a portion of a sample iscontacted with a binding surface so as to capture analyte on thesurface, the sample is removed, and the surface is contacted with alabeled analog of the analyte that binds to unoccupied binding sites onthe surface. The detection reagents may be present in a wash reagentused to displace sample material. Alternatively, the detection reagentsmay be added subsequent to the displacement of sample.

The measurement of analytes in the liquid fraction of a sample may becarried out using assay reagents supported on rough surfaces. Theroughness characteristics of the surfaces in such embodiments may beselected so that the surface structure prevents particles in a samplefrom contacting much of the surface while providing less of a barrier toanalytes and, optionally, soluble reagents that participate in the assay(such as detection binding reagents, e.g., detection antibodies). In oneembodiment, the ratio of the surface area accessible to an analyteand/or soluble reagent relative to the surface area accessible to aparticle in the sample is greater than two. In other embodiments, thisratio is greater than four, ten or fifty. In specific embodiments usefulin whole blood assays, the surface may be selected to provide a barrierto red blood cells but not to analyte and/or soluble reagents. By way ofexample, the surface may be selected so that the ratio of the surfacearea accessible to IgG molecules is two, four, ten or fifty timesgreater than the surface area accessible to red blood cells. The surfacemay be the surface of an inherently rough material or, alternatively, arough surface may be produced using, e.g., manufacturing techniquesknown in the art such as etching, micromolding, micromachining,electroforming, chemical vapor deposition, photolithography, etc. Thesurface may be formed from a composite material comprising particlesdistributed in a matrix, the exposed particles on the surface of thematerial providing the desired roughness characteristics. Suitablematerials include composites comprising carbon particles such asamorphous carbon particles, graphitic particles and carbon nanotubes.Optionally, the composite may be etched (e.g., by chemical or plasmaetching) to expose more particles and increase the surface roughness. Inone specific example, the surface is provided by a printed carbon ink.

According to another embodiment of the invention, the measurement ofanalytes in the liquid fraction of a sample (e.g., in the plasmafraction of a whole blood sample) is conducted under conditions in whichthere is no substantial depletion of analyte from the sample. Themeasurement may involve an assay reaction that reaches equilibriumwithout substantially depleting analyte from the sample. For example theamount and binding affinity of binding reagents in one or more bindingdomains may be chosen so that the binding of analytes in a sample tothese binding domains reaches equilibrium without binding more than 30%of any analyte (no more than 10%, no more than 20%, or, no more than 1%)to the corresponding binding domain. In one embodiment the amount of abinding reagent (in moles) present in a binding domain contacted with avolume of sample is not more than 0.1 V/K where V is the volume ofsample (in liters) and K is the affinity constant for the binding of thebinding reagent to analyte (in liters/mol).

According to another embodiment, the assay may be conducted withoutsignificant depletion of analyte by carrying out the assay in a kineticregime, i.e., by carrying out the assay reactions for a period of timewhich may be a pre-determined period of time) that is shorter than thetime required for the reaction to reach equilibrium. This time may beselected so that no more than 30%, 20%, 10% or 1% of an analyte isdepleted from the sample, e.g., by binding of the analyte to a bindingdomain. The time in certain embodiments is less than 15 minutes, lessthan 10 minutes, less than 5 minutes or less than 3 minutes. In oneembodiment, at least a portion of a sample is contacted with a surface,for example, with a surface containing a first binding partner of ananalyte of interest immobilized thereon, for a defined interval of timeshorter than the interval of time required to reach an equilibriumbetween the analyte immobilized on the surface and free in solution, incertain embodiments for a period of time shorter than 15 minutes,shorter than 10 minutes, shorter than 5 minutes, or shorter or equal tothree minutes.

According to another embodiment, measurement of analytes in the liquidfraction of a sample (e.g., the plasma fraction of whole blood) iscarried out in a solid phase assay format by flowing the sample over anassay surface, e.g., a surface with one or more binding domains. Incertain embodiments, the flow is carried out under conditions thatimprove mass transport of analyte to the assay surface or assay domainsdefined thereon. The sample may be passed over the surface underconditions of continuous flow. Alternatively, the sample may be movedback and forth over the surface (reciprocal flow) to increase thecontact time between a volume of sample and the assay surface. Duringperiods of flow, the flow may be turbulent or laminar. The flow rate incertain embodiments is selected to provide substantially laminar flow.The laminar nature of the flow can be characterized by the Reynold'snumber of the flow. The Reynold's number for the flow may be less than100 or less than 10. In one specific embodiment, the assay surfaces usedin a solid phase assays of the invention define, at least in part, theinterior surfaces of a flow cell through which the sample is passed. Theflow rates and flow cell dimensions may be chosen so as to providelaminar flow of sample through the flow cell. In certain embodiments,analyte is not substantially depleted from the sample during the assay.

According to another embodiment, the flow of sample over an assaysurface is controlled so as to focus the flow of particulates into flowpaths which minimize contact of the particulates with the surface, thusproviding a layer of low-particulate liquid over the surface. In oneexample, the flow conditions are selected to provide laminar flow overthe assay surface with the particulates in the sample restricted tofaster moving laminae of the flow-removed from the surface and, e.g.,located towards the middle of the flow path of sample through a flowcell (FIG. 1). In the case of blood samples, a plasma-rich layer can,therefore, be maintained directly adjacent the assay surface. Withoutbeing restricted to any particular physical theory or explanation,although blood is a non-Newtonian fluid, it is believed that under flowand high shear rates the red blood cells tend to separate fromaggregates, common at quiescent conditions, and align with the flow as aresult of cell stretching or deforming. (Boryczko K., et al., (2003) J.Mol. Model., 9:16-33; Pries, A. R, et. al. (1992) Am. J. Physiol. HeartCirc. Physiol. 263: H1770-H1778). This alignment also causes the bloodcells to move in layers sliding past clear layers of plasma. In certainembodiments, analyte is not substantially depleted from the sampleduring the assay.

For embodiments wherein a flowing fluid is involved, the fluid flow is,optionally, carried out under conditions that provide a flow rate thatis independent of viscosity for the range of samples that may beanalyzed. By way of example, a sample may be pumped (e.g., byapplication of pressure or vacuum to the sample or electro-osmotic flow,etc.) through a cell or conduit (which may be a flow cell with assaysurfaces or a separate conduit), the flow rate through the conduit maybe measured (e.g., through the use of capacitance, optical orconductometric sensors to measure the time the sample takes to move adefined distance through the cell or conduit) and the pressure or vacuummay be adjusted to adjust the flow rate to a predetermined desiredvalue.

According to another embodiment, measurement of analytes in the liquidfraction of a sample (e.g., the plasma fraction of whole blood) iscarried out in a solid phase assay format by introducing the sample intoan assay cell (e.g., by flowing the sample into a flow cell orintroducing the sample into an assay tube, etc.). The assay cell cancomprise one or more assay surfaces (e.g., surface with one or morebinding domains or other type of assay domains), the surfaces beingarranged within the assay cell such that they face sidewise or downwardsduring operation of the cell. By way of example, the binding surface maydefine, at least in part, the top surface an internal chamber of theassay cell. The sample may be introduced into the assay cell and held inthe cell under conditions which allow particles in the sample to settle(e.g., under no flow or low flow conditions) away from the assaysurface(s) and thus provide a plasma-rich layer near the assaysurface(s). The method may further comprise, after the settling hasoccurred, flowing the sample past the assay surface(s) under laminarflow (optionally, in a back and forth movement) so as to introduceconvectional mixing while maintaining a plasma-rich layer near the assaysurface(s). In certain embodiments, analyte is not substantiallydepleted from the sample during the assay.

The solid phase binding methods of the present invention may furthercomprise displacing the whole blood sample, for example, displacing byintroducing an assay buffer or a wash solution onto the surface, incertain embodiments with an assay buffer or a wash solution containing asecond binding reagent, for example, a second binding reagent labeledwith a label, prior to measuring or prior to generating an assay signal.Certain methods may further comprise an act of contacting a surface witha second binding reagent, for example, with a second binding reagentlabeled with a label, or, in certain embodiments, labeled with an ECLlabel after the whole blood sample was displaced from a surface.

Certain methods of the invention may, advantageously, be used to performrapid blood tests and may be especially suited to carrying out tests inpoint-of-care settings, in particular at point-of-care settings whereusers may not be trained to use or have access to equipment forseparating plasma or serum from blood or where the available bloodsamples (e.g., finger prick samples) are too small in volume forconvenient preparation of plasma or serum. In one embodiment, theinvention is a method for performing a rapid blood test, for example ata point-of-care setting, which can be completed within 30 minutes,(within 20 minutes, or within 10 minutes) comprising:

a) drawing a sample of blood from a patient to provide a whole bloodsample;

b) applying the whole blood sample to an assay module (for example, anassay plate or an assay cartridge) comprising a binding surface;

c) flowing the whole blood sample over the surface for a definedinterval of time to immobilize an amount of an analyte of interest on asurface; and

d) measuring an amount of an analyte on a surface, for example, bymeasuring an amount of a label on a surface;

e) determining a measure of the concentration of the analyte in bloodplasma, for example, by using a calibrator;

wherein the amount of analyte immobilized on a surface is substantiallyindependent of the hematocrit of the whole blood sample and thedetermining act is conducted without hematocrit correction.

Advantageously, certain methods of the present invention can beperformed by users with minimal training requirements.

The assay surfaces or domains used in certain assay methods of theinvention may be comprised in assay modules (e.g., assay cartridges,assay plates, etc.) having one or more assay cells (e.g., wells,compartments, chambers, conduits, flow cells, etc.). One embodiment ofthe invention employs an assay module with an assay surface having anarray of assay domains. In one specific example of this embodiment, thearray is a two dimensional array. In another specific example, the arrayis a one dimensional array that is aligned along the flow path of a flowcell in an assay cartridge.

Assay domains may be supported on a variety of different assay surfacematerials including, but not limited to plastics, ceramics, metals,glasses, composites and the like. In embodiments that employelectrochemical or electrode induced luminescence measurements (such aselectrochemiluminescence measurements), the assay surfaces may be chosenand configured so as to be capable of acting as electrodes. Surfacessuitable for use as electrodes include surfaces comprising materialscomprising elemental carbon, for example, a composite materialcontaining particulate carbon in a matrix, e.g., a carbon ink. The assaymodules may also include additional electrode surfaces to providecounter or reference electrodes. Assay modules, and in particular assaycartridges and cartridge readers, suitable for use in carrying outelectrochemiluminescence-based measurements using methods of theinvention are described in detail in copending U.S. patent applicationSer. No. 10/744,726, now issued as U.S. Pat. No. 7,497,997, herebyincorporated by reference.

One embodiment of the invention employs a cartridge that includes one ormore sample chambers, one or more detection chambers (e.g., detectionchambers adapted for use in ECL measurements as described below) and oneor more waste chambers for holding liquid wastes. The chambers areconnected in series by fluid conduits so that a sample introduced into asample chamber can be delivered into one or more detection chambers foranalysis and then passed into one or more waste chambers. This cartridgemay also include or more reagent chambers for storing liquid reagents,the reagent chambers connected via conduits to the other components soas to allow the introduction of the liquid reagents into specifiedsample or detection chambers. The cartridge may also include vent portsin fluidic communication with the sample, detection and/or wastechambers (directly or through vent conduits) so as to allow theequilibration of fluid in the chambers with the atmosphere or to allowfor the directed movement of fluid into or out of a specified chamber,e.g. in or out waste chambers to the detection chamber, by theapplication of positive or negative pressure.

The detection chambers may be adapted for carrying out a physicalmeasurement on the sample. The detection chamber may be connected to aninlet conduit. In certain embodiments, the detection chamber is alsoconnected to an outlet conduit and is arranged as a flow cell. Incertain embodiments, the flow along the flow cell during its operationis substantially laminar, with Reynolds numbers less than 1000, lessthan a 100, less than 10 and in certain embodiments less than 5. If themeasurement requires illumination or optical observation of the sample(e.g., as in measurements of light absorbance, photoluminescence,reflectance, chemiluminescence, electrochemiluminescence, lightscattering and the like) the detection chamber may have at least onetransparent wall arranged so as to allow the illumination and/orobservation. When employed in solid phase binding assays, the detectionchamber may comprise a surface (e.g., a wall of the chamber) that hasone or more binding reagents (e.g., antibodies, proteins, receptors,ligands, haptens, nucleic acids, etc.) immobilized thereon (e.g., anarray of immobilized binding reagents, such as an array of immobilizedantibodies and/or nucleic acids). In one such embodiment, the array is aone-dimensional array aligned along the flow path of a flow cell. Inanother such embodiment, at least two of the binding domains formed bythe binding reagents differ in specificity for analytes of interest.

In one embodiment, the detection chamber is an electrochemiluminescencedetection chamber, which may have one or more binding reagentsimmobilized on one or more electrodes. In one embodiment, the cartridgecomprises a working electrode having an array of binding reagentsimmobilized thereon. In another embodiment, the cartridge comprises anarray of independently controllable working electrodes each having abinding reagent immobilized thereon.

One embodiment of an electrochemiluminescence detection chamber is achamber having a fluid inlet and fluid outlet and a flow path betweenthe inlet and outlet.

An array of electrodes may be patterned on an internal surface of thechamber, for example, in a one dimensional array along the fluid flowpath. The internal chamber surface opposing the electrode array may belight-transmissive so as to allow for the detection of light generatedat the electrodes. One or more of the electrodes may comprise assayreagents, for example, a first binding reagent, immobilized on theelectrode. These assay domains may be used to carry out assay reactionswhich can be detected by using the electrode to induce an assaydependent signal, such as an electrochemical or, an electrode inducedluminescence signal, and detecting the signal. In certain embodiments,these assay reagents are arranged in one or more assay domains definedby apertures in a dielectric layer deposited on the electrode.

The cartridge may comprise additional reagents used in the assaymethods, e.g., binding reagents (such as antibodies, nucleic acids, etc.which may be labeled with a detectable label), agents that compete withan analyte for binding to a binding reagent, anticoagulants (e.g.,heparin, citrate, oxalate, EDTA, etc.), pH buffering components, salts,blocking agents (e.g., proteins that block non-specific binding and/orblock the binding of heterophile antibodies) and preservatives (e.g.,fluoride, iodoacetate, etc.). One or more of these reagents may beprovided on the cartridge in dry form. Such a cartridge may also provideliquid reagents such as wash buffers, extraction buffers, read buffers(e.g., ECL coreactant containing solutions for ECL-based assays).

An assay cartridge may contain active mechanical or electroniccomponents such as pumps, valves, sensors (e.g., light detectors),sources of electrical power and the like as needed, e.g., to move fluidsin the cartridge or to generate and/or detect assay signals. In oneembodiment of the invention, an assay cartridge has minimal or no activemechanical or electronic components. When carrying out an assay, such anassay cartridge may be introduced into a cartridge reader which providesthese functions. For example, the reader may have pumps, valves,heaters, sensors, etc. for providing fluids to the cartridge, verifyingthe presence of fluids and/or maintaining the fluids at an appropriatecontrolled temperature. The reader may be used to store and provideassay reagents, either onboard the reader itself or from separate assayreagent bottles or an assay reagent storage device. In one embodiment ofa cartridge reader, the reader moves fluids in a cartridge by applyingpositive or negative air pressure to ports on the cartridge, in certaincases, without the cartridge reader coming in contact with liquidsamples or reagents. The reader may also have sensors, such ascapacitive sensors or optical sensors, for monitoring and allowingprecise control of fluid movement through the cartridge.

A assay procedure using an assay module and assay reader may compriseinserting the cartridge in the reader, making the appropriateelectrical, fluidic and/or optical connections to the cartridge (makinguse of electrical, fluidic and/or optical connectors on the cartridgeand reader), and conducting an assay in the cartridge. A whole bloodsample may be introduced into the cartridge prior to inserting thecartridge in the reader. The assay may also involve adding one or moreassay reagents to the cartridge; for example, one or more assayreagents, such as binding reagents, are stored in the cartridge in a dryand/or wet form.

FIG. 2 is a schematic representation of cartridge 200, one embodiment ofa cartridge of the invention that incorporates many of the fluidicfeatures described above. This exemplary embodiment depicts a cartridgecomprising an electrode array of the invention. The skilled artisan,however, can readily adapt the fluidic components and design tocartridges employing other detection chamber designs and/or detectiontechnologies. Cartridge 200 comprises various compartments including asample chamber 220, assay reagent chamber 225, waste chambers 230 and231 and detection chambers 245 and 246 comprising electrode arrays 249 aand 249 b and electrode contacts 297 and 298. Also depicted in FIG. 2are fluid ports/vents 250-253 and 280 that may be utilized as fluidiccontrol points, vents for allowing a chamber to equilibrate withatmospheric pressure, ports for introducing air bubbles or slugs into afluid stream and/or as fluidic connections to a cartridge reader. FIG. 2also depicts a number of fluidic conduits (shown as lines connecting thevarious chambers) that establish a fluidic network that connects thevarious compartments and/or fluid ports/vents. The fluidic conduits maycomprise distribution points to distribute a fluid to two or morelocations/compartments in a cartridge and sharp angles that preventpassive fluid flow (ensuring that fluid movement past these featuresonly occurs during active movement of the fluids). Other fluidicfeatures that are shown in FIG. 2 include pill chambers/zones 290, 291for each of the read chambers.

Sample chamber 220 is a chamber defined within cartridge 200 that isadapted for receiving a sample, for example a liquid sample, to beanalyzed in the cartridge. Cartridge 200 may also include a sealableclosure for sealing sample introduction port 221. Reagent chamber 225 isa chamber adapted to hold a liquid reagent and includes a vent conduitlinked to vent port 250 and reagent conduit linked to the sampleconduit. Pill chambers/zones 290 and 291 hold dry reagents and arepositioned, respectively, in the fluidic pathway between sample port 220and detection chambers 245 and 246 so that liquid passing through thechamber/zones will reconstitute the dried reagents and carry theresulting solutions into the detection chambers. Reagent chamber 225,vent port 280 and/or pill chamber zones 290 and/or 291 may optionally beomitted.

Detection chambers 245 and 246 are adapted for carrying outelectrochemiluminescence-assays and comprise arrays of electrodes 249 aand 249 b having binding domains 248 a and 248 b comprising immobilizedbinding reagents. During operation, the cartridge may be held (e.g., bya cartridge reader) so that the surface having the binding domains facessubstantially upward, downward or sideways. Optionally, detectionchamber 246 is omitted. Detection chambers 245 and 246 are connected viawaste conduits to waste chambers 231 and 230. Waste chambers 230 and 231are chambers configured to hold excess or waste fluids and are alsoconnected, respectively, to vent port 252 via a vent conduit and ventport 251 via a vent conduit. The use of multiple waste chambersadvantageously allows fluid flow through the multiple chambers to becontrolled independently via the application of vacuum or pressure tothe waste chamber vent ports. Alternatively, only one waste chamber isused (e.g., waste chamber 230 is omitted and detection chambers 245 and246 are both connected to waste chamber 231).

FIG. 3A shows an exploded view of cartridge 300, one implementation ofcartridge 200 that comprises cartridge body 310 and cover layers 324,350, 320, 321 and 322 mated to the surfaces of cartridge body 310 eitherdirectly or through gasket layers such as gasket layers 330 and 331.Cartridge body 310 includes features (channels, grooves, wells,compartments, etc.) and may be prepared by injection molding of aplastic. The features are sealed to provide some of the chambers andconduits of the cartridge by applying the cover layers to the upper andlower portions of the cartridge body (either directly or through anintervening gasket layer). Specifically, detection chambers (such asdetection chambers 245 and 246 in FIG. 2) are formed by sealing coverlayer 350 (having patterned conductive layer 360 (which forms thepatterned electrode array 249 a and 249 b, shown in FIG. 2) andpatterned dielectric overlayer 365) to cartridge body 310 throughintervening gasket layer 331 made in certain embodiments, from doublesided adhesive tape). FIG. 3B shows the alignment of gasket layer 331with cover layer 350. The detection chamber's depth, length and widthare defined by cutouts 340 and 341 within the gasket layer. Holes inpatterned conductive layer 360 define an array of exposed electrodesurfaces in the detection chambers on which binding reagents areimmobilized so as to form one-dimensional arrays of binding domains inthe detection chambers.

In the embodiment shown in FIG. 3, the cartridge body further includeselectrical access regions 395 and 396 that, together with cutouts 370and 371 in gasket layer 331 allow electrical contact to be made withelectrode contacts 397, 398. Electrical access regions are cut-outs orholes in the cartridge body configured and arranged to be in alignmentwith the electrode contacts.

Certain features of invention are further illustrated by the followingexamples.

EXAMPLES

The following examples are illustrative of some of the methods andinstrumentation falling within the scope of the present invention. Theyare, of course, not to be considered in any way limitative of theinvention. Numerous changes and modifications can be made with respectto the invention by one of ordinary skill in the art without undueexperimentation.

Materials & Methods:

Pooled Cardiac Marker Stock Solution:

A pooled cardiac marker stock solution was prepared that containedmyoglobin, cardiac troponin I (TnI), CKMB, and cardiac troponin T (TnT)at 2547, 28.8, 225.5 and 39.4 ng/ml, respectively. Calibration standardswere prepared by diluting the pooled cardiac marker stock solution withoff-the-clot pooled human serum (Western States Plasma #HS-300)containing the additives 0.1% w/v 2-chloracetamide, 0.01% w/v2-methyl-4-isothiazolin-3-one, 2.0% w/v sucrose, 0.23% w/v tetrasodiumEDTA, 0.16% w/v N-acetyl-L-cysteine, and 0.085% w/v potassium ADP.

Spiked Blood Samples:

In several of the examples described below, a number of whole bloodsamples were prepared that differed in hematocrit value but hadequivalent concentrations of cardiac markers in their respective plasmafractions. That strategy used to make these test samples is illustratedin procedure described below.

Whole blood samples containing lithium heparin as an anticoagulant wereobtained from the Research Sample Bank (Pompano Beach, Fla.) anddetermined to be free of red blood cell lysis before use. A spiked wholeblood standard containing added cardiac markers was prepared bycombining 9500 ul of the whole blood (39.7% hematocrit) with 500 ul ofthe pooled cardiac marker stock solution and 100 ul of a solutioncontaining blocking agents (50 mg/mL bovine serum albumin and 5 mg/mLbovine IgG). The spiked blood standard (final hematocrit level of 37%)was mixed for 50 minutes in a gentle orbital motion on an OxisInstruments Nutator (Ivyland, Pa.). The spiked blood standard was usedto prepare additional whole blood samples having the same plasma levelsof the cardiac markers but different hematocrit levels.

To prepare a spiked sample with a hematocrit level of 56%, 1.500 mL ofthe spiked blood standard was spun gently on a StatSpin-RX (Norwood,Mass.) mini-centrifuge plasma separator and 0.500 mL plasma was removed.To prepare a spiked sample with hematocrit level of 47%, 1.500 mL of thespiked blood standard was spun gently and 0.300 mL plasma was removed.To prepare a spiked sample with a hematocrit level of 28%, 0.75 mL ofspiked blood standard was combined with 0.25 mL of plasma prepared bycentrifuging a separate aliquot of spiked blood standard. To preparehematocrit level of 19%, 0.75 mL of spiked blood standard was combinedwith 0.75 mL of plasma prepared by centrifuging a separate aliquot ofspiked blood standard. Finally, blood plasma prepared by centrifugingthe spiked blood standard was used for a 0 hematocrit sample.

All the spiked samples were mixed in a gentle orbital motion on an OxisInstruments Nutator (Ivyland, Pa.) for 10 minutes at room temperaturebefore use.

Cardiac Marker Antibodies:

Capture antibodies for myoglobin, TnT, CKMB, and TnI were biotinylatedwith biotin-LC-sulfo-NHS-ester (BioVeris Inc., Gaithersburg, Md.).Detection antibodies for the same analytes were labeled with Sulfo-TAGNHS Ester (Meso Scale Discovery, Gaithersburg, Md.), anelectrochemiluminescent label based on a sulfonated derivative ofruthenium-tris-bipyridine (compound 1 pictured below). Labeledantibodies were purified by size exclusion chromatography on SephadexG-25 or G-50 (Pharmacia Biosciences).

Cartridge Assays:

The assays were conducted using assay cartridges as shown in FIG. 3 andas described in more detail in U.S. Pat. No. 7,497,997. Cover layer 350was provided by a sheet of mylar having a layer of carbon ink formingelectrode arrays 360 screen printed thereon and a dielectric layer 365having apertures that defined the exposed electrode surfaces within thedetection chamber. Gasket layer 331 was provided for by double sticktape. The thickness of the double stick tape and the dimensions of theslots were selected so that the detection chambers were 3.175 mm wideand 0.127 mm high and several cm long. Capture antibodies against,myoglobin, TnT, TnI, CKMB and/or progesterone were immobilized on theexposed surfaces so that the surface defined by any one of the apertureshad capture antibody directed against only one analyte. Theimmobilization was carried out by microdispensing solutions containing60 ug/mL of an antibody on the exposed area (so that it spread to butnot past the boundary defined by the dielectric layer), allowing thesolution to dry on the electrode, and then blocking any uncoatedelectrode surface with a BSA solution. Capture antibodies wereimmobilized in the same pattern in the two detection chambers of theflow cells, allowing each measurement to be carried out in duplicate.

The assays were conducted in one- or two-step formats. Unless notedotherwise, the assays were carried out as described below. The one-stepassays were carried out by introducing the sample (premixed with labeleddetection reagents for the analytes being measured; antibodies forsandwich assays or analogs of analytes for competitive assays) into thecartridge. By applying vacuum/pressure to and/or venting the appropriatevent ports, sample was introduced into the detection chambers and movedback and forth through the chamber for a period of 4 minutes. The samplewas then washed from the detection chamber with a buffered solutioncontaining tripropylamine (MSD Read Buffer T, Meso Scale Diagnostics)and the detection chambers were left filled with the tripropylaminesolution. ECL was induced by applying an electrical voltage signal tothe electrode contacts (2-5 V over 5 seconds) and the resulting ECL wasimaged with a cooled CCD camera. Image analysis software was used toquantitate the ECL emitted from each element of the antibody array.

The two-step assays comprised introducing a sample into the cartridge(in the case of the two-step assay the sample was not premixed withdetection reagents), introducing the sample into the detection chambersand moving it back and forth in the chamber for 3 minutes. Washing thesample from the chamber, introducing a solution containing theappropriate detection reagents, and moving this solution back and forththrough the chamber for 1 minute. The detection reagents were thenwashed away with tripropylamine solution and ECL was generated andmeasured as described for the one-step assay.

Sample and/or detection reagent solutions were moved through thedetection chambers at a flow rate of 10 uL/min For the detection chamberdimensions used and the viscosity and density of the samples, this flowrate corresponds to a Reynold's number for the flow of roughly 3.2.

Example 1 Hematocrit-Independent One-Step Sandwich Immunoassay forPlasma Levels of Cardiac Markers in Whole Blood Samples

Six test samples having the same plasma levels of cardiac markers butdifferent hematocrit levels were prepared as described in Materials andMethods. Table 1 provides, for each of the samples, the hematocrit, theconcentration of each of the analytes in the blood sample (bloodconcentration) and the concentration of each of the analytes in theplasma fraction (plasma concentration).

TABLE 1 Blood Concentration Plasma Concentration (ng/mL) (ng/mL) SampleHematocrit (%) Myo TnI CKMB TnT Myo TnI CKMB TnT 1 0 200 2.3 18 3.1 2012.3 18 3.1 2 19 163 1.9 15 2.5 201 2.3 18 3.1 3 28 145 1.7 13 2.3 2012.3 18 3.1 4 37 127 1.4 11 2.2 201 2.3 18 3.1 5 47 107 1.2 9.5 1.6 2012.3 18 3.1 6 56 88 1.0 7.9 1.4 201 2.3 18 3.1

A solution containing detection antibodies against myoglobin, TnI, CKMBand TnT (3.3 μl) was mixed with 160 μl of each test sample. The finalantibody concentrations (in weight per total blood volume) were 2 μg/mlwith the exception of the anti-myoglobin antibody, which was 4 μg/ml.The mixtures were incubated for 2 minutes and 150 μl aliquots weretransferred into cartridges for measurement. The assays were conductedin one-step format. Two cartridges were used for each hematocrit levelgiving a total of four replicates per condition (2 per cartridge). Thereported signals are normalized by dividing the measured signal by theaverage signal for the condition that gave the lowest signal for eachassay.

FIG. 4 shows the normalized average signal and standard deviation foreach condition. The results show no dependence between the ECL signaland the hematocrit level. Samples with equivalent plasma levels ofcardiac markers gave roughly equivalent signals despite largedifferences in the hematocrit and the weight of analyte per volume ofwhole blood.

Example 2 Hematocrit-Independent One-Step Sandwich Immunoassay forPlasma Levels of Cardiac Markers in Whole Blood Samples

Example 1 compared the signals obtained for whole blood samples thatvaried in hematocrit but contained equal levels of analyte in theirplasma fractions. In this example, whole blood samples are analyzed thathave equal concentrations of analyte with respect to the total volume ofblood. The concentrations of analyte in the plasma should, therefore,vary as a function of the sample hematocrit.

The stock blood sample was prepared by combining 2282 μl of blood(hematocrit level of 42.5%) with 51 μl of a mixture of detectionantibodies for myoglobin, cTnT, cTnI, and CKMB at concentrations 193,97, 163, and 98 μg/ml, respectively and 33 μl of the BSA and bovine IgGblocking agent solution. Whole blood samples at four hematocrit levelswere prepared from stock blood sample as described in the Materials andMethods except that the blood stock contained no added analyte. Each ofthe whole blood samples was spiked with equal amounts of cardiac markersby addition of 26.6 μl of pooled cardiac marker stock solution to 0.5 mLof each hematocrit level. The samples were mixed for 1 hour beforetesting. Table 2 below lists, for each of the whole blood samples, thehematocrit, the plasma levels of the cardiac markers and theconcentration of the markers in the whole blood.

TABLE 2 Blood Concentration Plasma Concentration (ng/mL) (ng/mL) SampleHematocrit (%) Myo TnI CKMB TnT Myo TnI CKMB TnT 1 0 129 1.5 11 2.0 1291.5 11 2.0 2 20 129 1.5 11 2.0 161 1.8 14 2.5 3 39 129 1.5 11 2.0 2112.4 19 3.3 4 58 129 1.5 11 2.0 306 3.5 27 4.7

The samples were transferred to cartridges for measurement and analyzedusing a one step assay format. FIG. 5 shows the assay signals for eachsample (after normalizing by dividing the signal by the signal reportedfor the zero hematocrit sample). The signal increased with increasinghematocrit confirming that the assay is indicative of analyte levels inthe plasma fraction.

Comparative Example 3 One-Step Sandwich Immunoassay for Cardiac Markersin Whole Blood Samples; Incubation Under Static Conditions

Whole blood (2800 uL with a hematocrit of 45.7%) was spiked with 112 uLof the pooled cardiac marker solution and 30 uL of a solution containingdetection antibodies against myoglobin, CKMB and TnT (giving finalantibody concentrations of 14, 3 and 5 ug/mL, respectively) as well asBSA and bovine IgG. Four test samples having the same plasma levels ofcardiac markers but different hematocrit levels were prepared from thespiked whole blood by a procedure analogous to that described in theMaterials and Methods. The following table 3 provides, for each of thesamples, the hematocrit, the concentration of each of the analytes inthe blood sample (blood concentration) and the concentration of each ofthe analytes in the plasma fraction (plasma concentration).

TABLE 3 Blood Concentration Plasma Concentration (ng/mL) (ng/mL) SampleHematocrit (%) Myo CKMB TnT Myo CKMB TnT 1 0 170 15.1 2.6 172 15 2.7 221 136 11.9 2.1 172 15 2.7 3 43 98 8.7 1.5 172 15 2.7 4 57 74 6.5 1.1172 15 2.7

The samples were transferred to cartridges for measurement and analyzedusing a one step assay format with a 4 minutes static incubation(without moving the sample fluid over the immobilized captureantibodies). FIG. 6 shows the normalized signals for each sample typealong with the signal for a control experiment with mixing. The resultsshow that there is a dependence on hematocrit; the signal for a givenplasma level decreases with increasing hematocrit and the correspondingdecrease in the amount of analyte per volume of blood. In contrast,assays run while flowing samples over the capture antibodies showed nodependence on hematocrit (see, Example 1).

Comparative Example 4 One-Step Sandwich Immunoassay for Cardiac Markersin Whole Blood Samples; End-Point Assay

Whole blood (14250 uL with a hematocrit of 43.4%) was spiked with 600 uLof the pooled cardiac marker solution and 150 uL of the BSA/bovine IgGblocking solution. Four test samples having the same plasma levels ofcardiac markers but different hematocrit levels were prepared from thespiked whole blood by a procedure analogous to that described in theMaterials and Methods. The following table 4 provides, for each of thesamples, the hematocrit, the concentration of each of the analytes inthe blood sample (blood concentration) and the concentration of each ofthe analytes in the plasma fraction (plasma concentration).

TABLE 4 Blood Concentration Plasma Concentration (ng/mL) (ng/mL) SampleHematocrit (%) Myo CKMB TnT Myo CKMB TnT 1 0 173 15.3 2.7 173 15 2.7 221 137 12.0 2.1 173 15 2.7 3 41 102 9.0 1.6 173 15 2.7 4 62 66 5.8 1.0173 15 2.7

A solution containing the detection antibodies for myoglobin, CKMB andTnT (10 uL) was mixed with 150 uL of each sample to give finalconcentrations over the total sample volume of 20, 5, and 3 ug/mL forthe myoglobin, TnT and CKMB antibodies, respectively. An aliquot (150uL) of each mixture was transferred to a cartridge for measurement andthe assays were conducted using a one step assay format with a one hourincubation time (sufficient time for the binding reactions to proceed tonear completion). FIG. 7 shows the normalized signals for each sampletype and shows that there is a dependence on hematocrit; the signal fora given plasma level decreases with increasing hematocrit and with thecorresponding decrease in the total amount of analyte added to thecartridge. In contrast, no dependence on hematocrit was observed whenassays were run under the same conditions except that for using ashorter incubation time that only allowed a small fraction of theanalyte to bind to the capture antibodies (see, Example 1).

Example 5 Hematocrit-Independent Two-Step Sandwich Immunoassay forPlasma Levels of Cardiac Markers in Whole Blood Samples

Whole blood samples with equal plasma levels of cardiac markers butvarying hematocrits were prepared as in Example 4. The detectionantibody solution had 5 ug/mL of labeled anti-TnT and 3 ug/mL ofanti-CKMB. The detection antibody solution also contained anti-TnI andanti-myoglobin detection antibodies; the number of labels per antibodyfor these two antibodies was, however, too low to get a good signal inthe TnI and CKMB assays and these data are not presented.

The assays were conducted in two-step format. FIG. 8 shows thenormalized signal. As observed in the analogous one step assay (Example1), the signals were independent of hematocrit.

Example 6 Hematocrit-Independent Competitive Immunoassay for PlasmaLevels of Progesterone in Whole Blood Samples

Whole blood (3600 uL with a hematocrit of 45.7%) was spiked with 360 uLof a progesterone solution (80 ng/mL in horse serum) and 40 uL of 50mg/mL bovine IgG, 5 mg/mL BSA. Four test samples having the same plasmalevels of progesterone but different hematocrit levels were preparedfrom the spiked whole blood by a procedure analogous to that describedin the Materials and Methods. The following table 5 provides, for eachof the samples, the hematocrit, the concentration of progestoerone inthe blood sample (blood concentration) and the concentration ofprogesterone in the plasma fraction (plasma concentration).

TABLE 5 Blood Plasma Hematocrit Concentration (ng/mL) Concentration(ng/mL) Sample (%) Progesterone Progesterone 1 0 12.2 12.2 2 21 9.7 12.23 41 7.2 12.2 4 54 5.6 12.2

One step assays were carried out by adding progesterone labeled with anECL label (10 ul; 170 ng/ml) to 150 μl of sample and mixing for 3minutes prior to introduction of the mixture to a cartridge. Two-stepassays were carried out by incubating the samples (without labeledprogesterone) in the cartridge, washing and then introducing a 10 ng/mLsolution of the labeled antigen. FIG. 9 shows that the 2-stepcompetitive assay gives the same signal for samples with the same plasmalevel of progesterone at different hematocrit levels. In contrast, theresults in the one-step assay show a ˜2.4-fold greater signal for thehigh hematocrit (54%) sample relative to the plasma sample. This resultis consistent with the higher plasma concentration of labeled antigen(2.3-fold increase) in the high hematocrit sample.

While several embodiments of the invention have been described andillustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and structures for performing thefunctions and/or obtaining the results or advantages described herein,and each of such variations, modifications and improvements is deemed tobe within the scope of the present invention. More generally, thoseskilled in the art would readily appreciate that all parameters,dimensions, materials, configurations, etc. described herein are meantto be exemplary and that actual parameters, dimensions, materials,configurations, etc. will depend upon specific applications for whichthe teachings of the present invention are used. Those skilled in theart will recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described. Thepresent invention is directed to each individual feature, system,material and/or method described herein. In addition, any combination oftwo or more such features, systems, materials and/or methods, providedthat such features, systems, materials and/or methods are not mutuallyinconsistent, is included within the scope of the present invention.Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties. In cases where thepresent specification and a document incorporated by reference and/orreferred to herein include conflicting disclosure, and/or inconsistentuse of terminology, and/or the incorporated/referenced documents use ordefine terms differently than they are used or defined in the presentspecification, the present specification shall control.

1. A method for performing a rapid blood test for an analyte of interestin a whole blood sample comprising acts of: (a) drawing a sample ofblood from a patient to provide said whole blood sample; (b) applyingsaid whole blood sample to a cartridge, wherein said cartridge comprisesa binding surface within a flow cell; (c) flowing said whole bloodsample over said surface by a back and forth laminar flow of said sampleover said binding surface so that at least a portion of said samplecontacts said binding surface, wherein said sample is flowed over saidsurface for a defined interval of time to immobilize an amount of saidanalyte on said surface; (d) measuring said amount of said analyteimmobilized to said surface; (e) determining, from said amount, aconcentration value of said analyte in said sample that differs from theactual plasma concentration of said analyte in said sample by no morethan 20%; wherein said determining act does not include a hematocritcorrection.
 2. The method of claim 1, wherein the portion of the wholeblood sample that contacts the binding surface consists substantially ofplasma.
 3. The method of claim 1, wherein prior to applying said wholeblood sample to said cartridge, said whole blood sample has not beensubjected to a treatment effecting separation or partitioning of bloodcells from the plasma fraction of the whole blood sample.
 4. The methodof claim 1, wherein immobilization of said analyte is performed over aninterval of time which is less than 10 minutes.
 5. The method of claim1, further comprising calibrating said method using calibrator sampleswith known concentrations of said analyte.
 6. The method of claim 5,wherein said calibrator samples are substantially free of red bloodcells.
 7. The method of claim 1, wherein said measuring is conducted ina sandwich assay format or in a competitive assay format.
 8. The methodof claim 1, further comprising contacting said sample with a bridgingreagent that binds said binding reagent and said analyte.
 9. The methodof claim 1, further comprising binding said analyte of interest to alabeled binding reagent.
 10. The method of claim 1, further comprisingcontacting said surface with a labeled analog of said analyte.
 11. Themethod of claim 9, wherein a label of said labeled binding reagent isselected from a group consisting of an electrochemiluminescent (ECL)label, luminescent label, fluorescent label, phosphorescent label,radioactive label, or light scattering label and combinations thereof.12. The method of claim 10, wherein a label of said labeled analog isselected from a group consisting of ECL label, luminescent label,fluorescent label, phosphorescent label, radioactive label, or lightscattering label and combinations thereof.
 13. The method of claim 1,wherein said binding surface is an electrode surface.
 14. The method ofclaim 1, wherein said surface displays a surface area accessible to saidanalyte that is at least two-fold larger than the surface areaaccessible to red blood cells.
 15. The method of claim 1, wherein saidflow has a Reynold's number of less than
 100. 16. The method of claim 1,wherein said flow provides a plasma-rich layer adjacent to said surface.17. The method of claim 1, further comprising displacing said wholeblood sample from said surface prior to said measuring said amount ofsaid analyte on said surface.
 18. The method of claim 1, furthercomprising displacing said whole blood sample from said surface prior togenerating said assay signal.
 19. The method of claim 1, wherein saidwhole blood sample is displaced from said surface by introducing a washsolution.
 20. The method of claim 19, wherein after displacing saidwhole blood sample, the surface is further contacted with a solutioncontaining a labeled binding reagent.
 21. The method of claim 1, whereinsaid whole blood sample is undiluted.
 22. The method of claim 1, furthercomprising adding anticoagulants to said whole blood sample.
 23. Themethod of claim 1, wherein said act of applying said sample to saidcartridge comprises contacting at least a portion of the sample with aplurality of binding domains on one or more binding surfaces within saidflow cell, said binding domains having different specificity foranalytes of interest.
 24. The method of claim 23, wherein one or moreadditional analytes are measured in said sample.
 25. The method of claim1, wherein said binding surface faces downward or sidewise during saidimmobilization such that red blood cells in said sample settle away fromsaid binding surface.